Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
1 Composed for Protocol Collection @ OXPHOS International. Contributed by Dr. . Title of the Protocol Modified after: . . Reagents: Prepare 0.1 M Tris¯HCl, pH 8.0; Prepare 1 mM DTNB solution, 4 mg of 5,5'-dithiobis-(2-nitrobenzoic acid) per10 ml of 0.1 M Tris-HCl (pH 8.0) (fresh daily); Procedure: In a total volume of 1 ml, the assay medium is composed of 0.1 mM DTNB (100 l), 0.36 mM acetyl-CoA (30 l), 0.05 - 0.15 mg of mitochondrial protein, and 0.5 mM OAA (50 l), in Tris-HCL buffer (~0.8 ml). Perform assay at 25°C; Set spectrophotometer to 412 nm, blank by air; Dissolve (mitochondria) sample in 0.1% Triton X-100; Add mitochondria to the mixture of DTNB and Acetyl-CoA, record absorbance changes for 1 min; Add 50 l OAA, record absorbance increase for 2 min; Calculate specific activity as mU/mg of mitochondrial protein; for that, subtract the rate of absorbance changes before OAA addition (if any) from that after OAA addition and divide it by the extinction coefficient for TNB, EmM=14.15 mM-1 cm-1 and by mitochondria protein content. 1 mU of enzyme catalyzes the reduction of 1 nmol of DTNB per min. Comments: Supplement: Copyright status: Information available from the OXPHOS International Web pages is within the public domain; it may be freely distributed and copied. This site also contains resources which may incorporate material contributed or licensed by individuals, companies, or organizations that may be protected by U.S. and foreign copyright laws. All persons reproducing, redistributing, or making commercial use of this information are expected to adhere to the terms and conditions asserted by the copyright holder. Transmission or reproduction of protected items beyond that allowed by fair use as defined in the copyright laws requires the written permission of the copyright owners. Liability: For documents and software available from this server, the OXPHOS International and individuals who contributed the content materials do not warrant or assume any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed. Conditions of Use: This site is maintained by the OXPHOS International and is protected by various provisions. For site security purposes, as well as to ensure that this service remains available to all users, we use software programs to monitor traffic and to identify unauthorized attempts to upload or change information or otherwise cause damage. In the event of authorized law enforcement investigations and pursuant to any required legal process, information from these sources may be used to help identify an individual. Revised: March 05, 2005.